ATP7A Orchestrates Tumor Progression and Cuproptosis in Hepatocellular Carcinoma via the LINC02038‐miR‐506‐3p Regulatory Circuit

ABSTRACT

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options, underscoring the urgent need for novel therapeutic targets. The copper transporter ATP7A has been implicated in cancer, but its precise role and regulatory mechanisms in HCC pathogenesis remain poorly understood. ATP7A expression was analyzed using public databases and validated in clinical HCC tissues and cell lines via qPCR and western blot. Functional assays were performed following ATP7A knockdown. Sensitivity to cuproptosis was assessed by measuring intracellular copper/ROS levels and IC50 of elesclomol‐Cu. The upstream regulatory axis was identified through bioinformatic prediction and verified by dual‐luciferase reporter and rescue experiments. The role of the LINC02038/miR‐506‐3p/ATP7A axis was further confirmed both in vitro and in a xenograft mouse model. ATP7A was significantly upregulated in HCC tissues and cell lines. ATP7A knockdown markedly inhibited HCC cell proliferation, migration, and invasion, while simultaneously sensitizing cells to cuproptosis. Mechanistically, LINC02038, which was highly expressed in HCC, functioned as a competing endogenous RNA (ceRNA) to sponge tumor‐suppressive miR‐506‐3p, thereby upregulating ATP7A expression. This LINC02038/miR‐506‐3p/ATP7A axis was demonstrated to coordinately regulate malignant phenotypes and the expression of key cuproptosis‐related proteins (DLAT, FDX1, LIPT1). In vivo, silencing LINC02038 effectively suppressed tumor growth and recapitulated the molecular alterations of this axis and cuproptosis regulators. Our findings reveal that the LINC02038/miR‐506‐3p/ATP7A axis played a crucial oncogenic role in HCC by driving tumor progression and modulating cuproptosis. This axis represents a promising prognostic biomarker and a potential therapeutic target for HCC intervention.

In hepatocellular carcinoma (HCC), upregulated LINC02038 acts as a competing endogenous RNA (ceRNA) that sponges tumor‐suppressive miR‐506‐3p, leading to derepression of ATP7A expression. Elevated ATP7A subsequently promotes HCC cell proliferation, migration, and invasion, while simultaneously modulating cuproptosis sensitivity.

1. Introduction

Hepatocellular carcinoma (HCC) ranks as the sixth most common malignancy worldwide and is typically associated with poor prognosis. According to World Health Organization projections, annual deaths attributable to HCC are predicted to surpass one million by 2030. While early diagnosis and intervention substantially improve patient outcomes [1, 2], effective therapies for advanced HCC remain limited, with most patients receiving only palliative treatments such as chemoembolization. Furthermore, current treatment strategies are often suboptimal due to issues including pronounced tumor heterogeneity and drug resistance [3]. Consequently, elucidating the molecular mechanisms driving HCC development and identifying novel biomarkers for prognostic assessment are essential for enhancing survival and quality of life in HCC patients.

Copper, an essential trace element in humans, participates in numerous critical biochemical processes [4]. While excessive copper ions exert cellular toxicity and disrupt redox homeostasis [5], elevated serum copper levels are frequently observed in cancer patients and correlate with disease progression and treatment response [6]. In recent years, cuproptosis—a novel form of cell death—has attracted increasing attention. This mechanism involves copper ions binding to lipid‐acylated components within the mitochondrial tricarboxylic acid cycle, leading to aggregation of lipid‐acylated proteins, depletion of iron‐sulfur cluster proteins, and ultimately triggering proteotoxic stress that induces cell death [7]. Cuproptosis plays a significant role in HCC. For instance, the copper‐dependent enzyme lysyl oxidase (LOX) shows markedly increased expression in HCC tissues, where it promotes collagen cross‐linking to enhance extracellular matrix stability, thereby supporting tumor growth and metastasis [8]. Zhang et al. [9] identified six cuproptosis‑related long non‐coding RNAs (AC138904.1, AC099329.2, DEPDC1‐AS1, GIHCG, AC145343.1, and DNMBP‐AS1) that are differentially expressed between tumor and normal tissues and closely associated with patient prognosis. Furthermore, studies reveal that MELK regulates the cuproptosis‑related protein DLAT via the PI3K/mTOR pathway to sustain mitochondrial function [10], while copper‐mediated SEC. 14L3 promotes cuproptosis through the ERK/YY1/FDX1 signaling axis, thereby suppressing HCC progression [11]. Thus, cuproptosis, as a newly identified cell death modality, holds important potential value for liver cancer therapy.

ATP7A gene is localized to the X chromosome region q12‑q13.3, encoding a copper‑transporting P‑type ATPase. This enzyme hydrolyzes ATP and utilizes released energy to facilitate transmembrane copper transport and efflux, thereby playing an essential role in maintaining intracellular copper ion homeostasis [12]. As a central regulator of cellular copper balance, ATP7A dysfunction is closely associated with tumor progression. Within cells, the copper chaperone ATOX1 constitutes a key component of the CTR1‑ATOX1‑ATP7A/ATP7B signaling axis [13]. By interacting with other proteins that regulate intracellular cuprous ions (Cu⁺) levels, ATOX1 participates in modulating Cu⁺ uptake, efflux, and subcellular distribution, thereby influencing the balance between cell survival and death [14]. Recent studies indicate that targeting ATP7A can induce cuproptosis. For example, in lung cancer, elesclomol promotes ubiquitin‑mediated degradation of ATP7A via upregulation of OSTM1, leading to intracellular copper accumulation and ultimately cuproptosis [15]. In HCC, ATP7A is frequently overexpressed and considered a potential target for immunotherapy and targeted therapy [16]. However, current understanding of the precise mechanisms by which ATP7A operates within cuproptosis pathways and its potential applications in HCC treatment remains limited and still in early stages. Therefore, further exploration into how ATP7A regulates cuproptosis in HCC holds significant value for developing novel therapeutic strategies.

This study aims to investigate the specific regulatory role of ATP7A in hepatocellular carcinoma (HCC) development, with a focus on elucidating how it mediates HCC biological behavior by influencing cuproptosis. Furthermore, we seek to uncover upstream molecular mechanisms governing ATP7A expression, thereby providing novel theoretical insights into dysregulated copper homeostasis in HCC.

2. Materials and Methods

2.1. Clinical Sample

This study enrolled 36 patients with HCC who underwent surgical resection. Both tumor tissues and adjacent non‑tumorous tissues (3 cm from the tumor margin) were collected for subsequent analyses. All participants had complete clinical records, no history of other malignancies, and were treatment‑naive to chemotherapy or radiotherapy. The study protocol was approved by the institutional Ethics Committee. Resected tissue samples were stored at −80°C until further experimental analysis.

2.2. Cell Culture

The HCC cell lines LM3, Huh7, Hep3B, and MHCC‐97H, along with the normal human hepatocyte cell line LO2, were obtained from the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI‐1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin, and maintained in a humidified incubator at 37°C with 5% CO₂. Cells were harvested during the logarithmic growth phase for subsequent experiments.

2.3. Cell Transfection

HCC cells were seeded in 6‐well plates at a density of approximately 100,000 cells per well and cultured for 12 h at 37°C. Thereafter, the old medium was removed. Transfection was performed using Lipofectamine 2000 (Beyotime) according to the manufacturer's instructions, with the reagent being mixed with respective DNA or RNA constructs. The final concentration of each transfection mixture was adjusted to 100 nmol/L, gently mixed, and then added dropwise to the corresponding wells. After 6 h of incubation, the medium was replaced with fresh complete medium, and the cells were further cultured for 24 h to allow efficient uptake of the transfection complexes and stabilization of gene expression. The primer sequences were listed in Table S1.

Lentiviral particles expressing sh‑LINC02038 were purchased from Shanghai GeneChem Co. Ltd. Prior to infection, the cell culture medium was replaced with fresh serum‑free medium. A preliminary experiment was performed according to the manufacturer's instructions to determine the optimal multiplicity of infection (MOI), which was set as 20 in this study. The required volume of virus was calculated using the formula: virus volume = (MOI × cell number)/viral titer. During infection, 40 μL of infection enhancer was added per well to improve transduction efficiency. After 12–16 h of incubation, the medium was replaced with complete medium. At 96 h post‑infection, transduction efficiency was evaluated under a fluorescence microscope, and puromycin selection was subsequently applied to obtain stable cell lines.

2.4. qRT‐PCR

Total RNA was isolated from both clinical specimens (HCC and adjacent normal tissues) and cultured HCC cells with TRIzol reagent. Following quantification and quality assessment, the RNA was reverse‐transcribed into cDNA using a commercial kit. Quantitative real‐time PCR (qRT‐PCR) was then conducted with SYBR Green Master Mix, gene‐specific primers, and the synthesized cDNA as template. The expression levels of LINC02038, miR‐506‐3p, ATP7A, along with the reference genes GAPDH and U6, were amplified and detected on a qRT‐PCR system. The relative expression of each target was finally determined based on recorded cycle threshold (Ct) values and calculated via the 2^−ΔΔCt method. The primer sequences were listed in Table S1.

2.5. CCK8

HCC cells were seeded into 96‐well plates and maintained at 37°C in a 5% CO₂ atmosphere. After 48 h of culture, 10 μL of CCK‑8 reagent was added to each well to assess cellular metabolic activity. The plates were then returned to the incubator and allowed to react for 2, 4, 6, or 8 h. Finally, the absorbance (OD) at 450 nm was measured for each well using a microplate reader.

2.6. Colony Formation Assay

HCC cells were seeded at a density of 200 cells per well in 6‐well plates and cultured under standard conditions (37°C, 5% CO₂) for 14 days. Following incubation, the culture medium was removed, and the cells were gently washed twice with PBS. The cells were then fixed with 5 mL of 100% methanol per well for 15 min at room temperature and subsequently stained with 1 mL of 0.1% crystal violet for 15 min at room temperature. After rinsing and air‐drying, the number of colonies was counted under a microscope.

2.7. Cell Migration and Invasion Assay

HCC cells were trypsinized, resuspended in serum‐free RPMI‐1640 medium, and 200 μL of the cell suspension (approximately 2 × 10⁵ cells) was carefully transferred to the upper chamber of a Transwell insert. Depending on the experimental design, the membrane of the upper chamber was either pre‐coated with Matrigel (for invasion assay) or left uncoated (for migration assay). The lower chamber was filled with 600 μL of medium containing 10% FBS as a chemoattractant. After incubation at 37°C for 24 h, cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. To quantify migratory or invasive ability, cells in five randomly selected fields per membrane were counted under a microscope, and the average number per field was calculated.

2.8. Western Blot Analysis

After transfection, cells were collected and lysed in RIPA buffer. The resulting protein lysates were mixed with 2× protein loading buffer at a 1:1 ratio and then boiled for 5 min. After cooling, 30 μg of protein per sample was subjected to SDS‐PAGE and subsequently transferred onto a PVDF membrane using a standard wet transfer system. The membrane was blocked with 5% non‐fat milk for 2 h at 37°C, followed by incubation with primary antibodies (1:1000 dilution) for 2 h at 37°C. After washing, the membrane was incubated with corresponding secondary antibodies (1:1000 dilution) for 1 h at 37°C. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate and captured with a gel imaging system. GAPDH was used as the internal control for normalization and quantitative analysis of target protein expression.

2.9. Measurement of Intracellular Copper and ROS Levels

HCC cells in logarithmic growth phase were seeded and cultured to appropriate density, then treated with elesclomol‑Cu complex (prepared at a 1:1 ratio; final concentration 200 nM) for 24 h. After incubation, cells were collected and intracellular copper concentration was measured following instructions of the copper assay kit.

Intracellular ROS levels were determined by flow cytometry. Cells were loaded with dichloro‑dihydro‑fluorescein diacetate (DCFH‑DA) probe and incubated in dark for 2 h. Fluorescence intensity, reflecting relative ROS content, was then detected using a flow cytometer.

2.10. IC50

HCC cells were seeded in 96‑well plates at 3 × 10⁴ cells per well. Following 24‑h incubation for cell attachment, elesclomol‑Cu (1:1) was added to the wells. After another 24‑h incubation, 100 μL of complete medium containing 10% CCK‑8 reagent was added to each well. Plates were incubated at 37°C for 2 h, and absorbance at 450 nm was measured using a microplate reader. The half‑maximal inhibitory concentration (IC₅₀) was then calculated.

2.11. Subcellular Fractionation and Localization

The cytoplasmic and nuclear fractions of HCC cells were isolated using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen, Belmont). The expression levels of LINC02038 in the nucleus and cytoplasm were then quantified by qRT‑PCR. GAPDH and U6 served as cytoplasmic and nuclear markers, respectively.

2.12. RIP Assays

Cells were centrifuged at 1000 × g at 4°C and then lysed in 500 µL RIP buffer (Millipore, Sigma). From the lysate, 100 µL aliquots were incubated with either 5 µL normal IgG or 5 µL anti‑Ago2 antibody (1:1000, Abcam). The Magna RIP RNA‑Binding Protein Immunoprecipitation Kit (Sigma, Millipore) was used following the manufacturer's protocol. Finally, RT‑qPCR was performed to evaluate the enrichment levels of LINC02038 on the beads.

2.13. Dual‐Luciferase Reporter Assay

To validate the targeting relationship between LINC02038 and miR‐506‐3p, as well as between miR‐506‐3p and ATP7A, a dual‑luciferase reporter assay was performed. Wild‑type (WT) and mutant (MUT) fragments of LINC02038 or the 3′‑UTR of ATP7A were cloned into the pmiRGLO vector, generating the reporter plasmids WT‑LINC02038, MUT‑LINC02038, WT‑ATP7A, and MUT‑ATP7A. These plasmids were co‑transfected with either the miR‑506‑3p mimic or a negative control miRNA (miR‑NC) into cells using Lipofectamine 2000, following the manufacturer's protocol. After 48 h, luciferase activity was measured using the Dual‑Luciferase Reporter Assay System (Promega).

2.14. Xenograft Tumor Experiment

For the xenograft tumor model, cells from the sh‑NC and sh‑LINC02038 groups were resuspended at a density of 2 × 10⁷ cells/mL. Each nude mouse received a subcutaneous injection of 200 µL of cell suspension (containing 4 × 10⁶ cells) into the right flank, with six mice per group. Tumor volume was measured weekly and calculated using the formula: volume = (length × width²)/2. After 4 weeks, the mice were euthanized via cervical dislocation under anesthesia. The tumors were carefully excised, weighed, and divided for different purposes: one portion was snap‑frozen and stored at −80°C for subsequent total RNA extraction and detection of LINC02038, miR‑506‑3p, and ATP7A expression; another portion was fixed in 10% formaldehyde and also stored at −80°C for further analysis.

2.15. Immunohistochemical (IHC) Assays

Tumor tissues were fixed in 4% glutaraldehyde at 4°C for 24 h, dehydrated through graded ethanol, and embedded in paraffin. Sections (4 μm) were cut, then treated with 3% H₂O₂ for 10 min. Antigen retrieval was performed in citrate buffer at 95°C for 15 min. The sections were separately incubated overnight at 4°C with primary antibodies against Ki‑67 (1:400) and ATP7A (1:500), followed by exposure to HRP‑conjugated secondary antibody at 37°C for 60 min. After DAB development, sections were counterstained with hematoxylin for 3 min and imaged under a light microscope (Olympus).

2.16. Statistical Analysis

Data analysis and graph generation were performed using GraphPad Prism version 8.0. Normally distributed continuous data are presented as the mean ± standard deviation (SD). Comparisons among multiple groups were evaluated by one‐way analysis of variance (ANOVA). The correlation between LINC02038, miR‐506‐3p, and ATP7A was analyzed using Pearson's correlation coefficient. p < 0.05 was considered statistically significant.

3. Results

3.1. ATP7A Was Upregulated Across HCC and Associated With Poor Prognosis

We first analyzed the expression pattern of ATP7A across a pan‑cancer spectrum and observed dysregulated expression in most cancer types (Figure 1A). Mining of the TCGA database further revealed that ATP7A expression was significantly higher in tumor tissues compared with matched normal tissues (Figure 1B). Its upregulation was most pronounced in patients with Stage 2 and Stage 3 disease (Figure 1C), and elevated ATP7A levels were closely associated with poor prognosis (Figure 1D). To validate these findings, we collected 36 pairs of HCC tissues and paired adjacent non‑tumor samples from patients treated at our hospital. Experimental results demonstrated that both ATP7A mRNA and protein levels were markedly upregulated in HCC tissues (Figure 1E,F), consistent with the public database. Furthermore, ATP7A expression was examined in four different HCC cell lines, all of which showed increased expression (Figure 1G). The LM3 and MHCC97H cell lines with the highest ATP7A expression levels were selected for subsequent functional inhibition experiments, whereas the Huh7 cell line with lower expression levels was used for overexpression experiments. These findings indicated ATP7A showed promise as a molecular marker for HCC progression assessment.

Figure 1.

Cuproptosis‐related gene ATP7A was highly expressed in HCC and associated with a poor prognosis. A. ATP7A expression analysis in pan‐cancer via Timer database. B, C. Correlation analysis between high expression of ATP7A and patient stage via UALCAN database. D. The expression of ATP7A in HCC tissues (n = 36) was detected via qRT‐PCR. E. Through Kaplan‐Meier survival analysis, the prognostic value of ATP7A expression was evaluated in HCC patients. F. The protein expression of ATP7A in HCC tissues was detected via western blot. G. The mRNA expression of ATP7A in HCC cells and normal cells was detected via qRT‐PCR. *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. ATP7A Knockdown Impaired HCC Malignancy and Sensitizes Cells to Cuproptosis

Based on the marked overexpression of ATP7A in HCC cells, we conducted transient knockdown using two distinct siRNAs targeting ATP7A. Both si‐ATP7A#1 and si‐ATP7A#2 effectively suppressed both ATP7A mRNA and protein expression (Figure 2A,B), with si‐ATP7A#1 demonstrating stronger inhibition. Phenotypic assays showed that ATP7A knockdown repressed HCC cell proliferation (Figure 2C,D). Transwell experiments further revealed that ATP7A depletion significantly impaired the migratory (Figure 2E) and invasive (Figure 2F) capacities in LM3 and MHCC‐97H cells. Given prior reports linking ATP7A to copper homeostasis and cuproptosis in other tumors, we explored its role in copper‐induced cell death within HCC. After 24‐h elesclomol‐Cu treatment, ATP7A knockdown substantially elevated intracellular Cu concentration and ROS levels (Figure 2G,H). Moreover, measuring the IC50 of elesclomol‐Cu in knockdown cells indicated a clear decrease (Figure 2I), reflecting heightened sensitivity to copper‐induced death. Subsequently, a stable ATP7A‐overexpressing cell line (oe‐ATP7A) was established in Huh7 cells with low endogenous ATP7A expression. qRT‑PCR and Western blotting confirmed significant upregulation of ATP7A mRNA and protein levels (Figure S1A,B). Functional assays showed that ATP7A overexpression markedly enhanced the proliferation, migration, and invasion of Huh7 cells (Figure S1C,E). Following treatment with elesclomol‑Cu for 24 h, elesclomol‑Cu treatment significantly decreased intracellular copper concentration and ROS levels, while markedly increasing the IC50 value in oe‐ATP7A cells (Figure S1F–H). Together, these findings demonstrated that ATP7A promoted HCC cell proliferation, migration, and invasion while also modulating cellular cuproptosis.

Figure 2.

ATP7A regulated the malignant progression and cuproptosis of HCC cells. A, B. The ATP7A expression was detected in HCC cells after transfecting with si‐ATP7A#1, si‐ATP7A#2 via qRT‐PCR and western blot. C, D. EdU and Colony formation were employed to detect cell viability and growth. E, F. Transwell was employed to detect cell migration and invasion. G. The intracellular copper levels in si‐NC and si‐ATP7A cells treated with elesclomol‐Cu (1:1 ratio) for 24 h. H. ROS levels in si‐NC and si‐ATP7A cells treated with elesclomol‐Cu (1:1 ratio) for 24 h. I. The IC50 of elesclomol‐Cu in si‐NC and si‐ATP7A cells were determined using the CCK‐8 assay. **p < 0.01, ***p < 0.001.

3.3. LINC02038 Acts as a ceRNA to Regulate ATP7A Expression via miR‐506‐3P

To investigate the upstream regulatory mechanism of ATP7A in HCC, we predicted potential miRNAs targeting ATP7A using ENCORI and TargetScan databases. Intersecting results from both platforms identified four candidate miRNAs: hsa‐miR‐223‐3p, hsa‐miR‐495‐3p, hsa‐miR‐506‐3p, and hsa‐miR‐5688 (Figure 3A). Subsequent validation of their expression levels in HCC cell lines revealed that only miR‐506‐3p showed significantly low expression in both LM3 and MHCC‐97H cells (Figure 3B). Bioinformatic analysis indicated seven potential binding sites for miR‐506‐3p within the ATP7A region (Figure 3C). We further predicted upstream lncRNAs for miR‐506‐3p by integrating data from the ENCORI database and reported cuproptosis‐related lncRNAs (CRLs). Three candidate lncRNAs were identified: EIF2AK3‐DT, LINC02038, and TMEM147‐AS1 (Figure 3D). Expression validation demonstrated that only LINC02038 was significantly upregulated in HCC cells, while the other two showed no notable change (Figure 3E). Sequence analysis suggested 11 potential binding sites complementary to miR‐506‐3p within LINC02038 (Figure 3F). To investigate whether LINC02038 directly interacted with miR‐506‐3p, RIP assays were performed using an AGO2 antibody in HCC cells. The results showed that LINC02038 was significantly enriched (Figure 3G), indicating a direct binding between LINC02038 and miR‐506‐3p. To further validate the targeting relationships, we constructed wild‐type (WT) and mutant (MUT) luciferase reporter vectors for both LINC02038 and ATP7A, which were co‑transfected with either a miR‐506‐3p mimic or a negative control (miR‐NC). Dual‑luciferase reporter assays demonstrated that the miR‐506‐3p mimic significantly reduced the luciferase activity of the LINC02038‑WT and ATP7A‑WT reporters, but had no effect on their mutant counterparts (Figure 3H,I). We further assessed the subcellular distribution of LINC02038 in HCC cells, which revealed a predominant cytoplasmic localization (Figure 3J). Moreover, LINC02038 was markedly upregulated in HCC tissues, whereas miR‐506‐3p expression was substantially downregulated (Figure 3K). Correlation analyses further demonstrated negative associations of both LINC02038 and ATP7A with miR‐506‐3p (Figure 3L). Additionally, we examined their regulatory interactions at the cellular level. Overexpressing miR‐506‐3p markedly suppressed ATP7A expression (Figure 3M). Conversely, knocking down LINC02038 also reduced ATP7A levels, an effect partially reversed by miR‐506‐3p inhibitor (Figure 3N). Collectively, these results suggest that LINC02038 may function as a competing endogenous RNA that sequesterd miR‐506‐3p, thereby relieving its repression on ATP7A and contributing to malignant phenotypes in HCC cells.

Figure 3.

The ATP7A‐associated ceRNA regulatory network was constructed in HCC cells. A. Prediction of upstream miRNAs of ATP7A based on ENCORI and Targetscan. B. The predicted miRNAs expression was detected in HCC cells. C. The binding sites of ATP7A and miR‐506‐3p. D. Prediction of upstream lncRNAs of miR‐506‐3p based on ENCORI and CRLs (cuproptosis‐related lncRNAs). E. The predicted lncRNAs expression was detected in HCC cells. F. The binding sites of miR‐506‐3p and LINC02038. G, H. Dual‐luciferase reporter assay was applied to confirm the binding of LINC02038/ATP7A and miR‐506‐3p. I. RIP assays were used to examine LINC02038 enrichment in HCC cells co‐transfected with LINC0203 and miR‐506‐3p mimic. J. Nuclear‑cytoplasmic fractionation assays were conducted to determine the subcellular localization of LINC02038 in HCC cells. K The expression levels of LINC02038 and miR‐506‐3p in HCC tissues (n = 36) were measured by qRT‑PCR. (L) Pearson's correlation coefficient was used to analyze the correlations among LINC02038, miR‐506‐3p, and ATP7A in HCC tissues. M. ATP7A mRNA expression in HCC cells transfected with miR‐NC and miR‐506‐3p was detected via qRT‐PCR. N. ATP7A mRNA expression in HCC cells was detected via qRT‐PCR after transfecting with sh‐NC+anti‐miR‐NC, sh‐LINC02038+anti‐miR‐NC, sh‐LINC02038+anti‐miR‐506‐3p. ***p < 0.001.

3.4. LINC02038/miR‐506‐3p/ATP7A Axis Regulates HCC Malignancy and Cuproptosis

To further validate the functional role of the LINC02038/miR‐506‐3p/ATP7A axis in HCC, we conducted systematic functional rescue experiments. Knocking down LINC02038 in LM3 and MHCC‐97H cells significantly suppressed cell viability, proliferation, migration, and invasion (Figure 4A,D). These inhibitory phenotypes were partially rescued by co‐transfection with either a miR‐506‐3p inhibitor (anti‐miR‐506‐3p) or an ATP7A overexpression plasmid (oe‐ATP7A), evidenced by restored cell viability in CCK‐8 assays, increased colony formation, and elevated numbers of migrating and invading cells in Transwell assays. Regarding cuproptosis‐related functions, LINC02038 knockdown markedly increased intracellular Cu concentration and ROS levels, effects that were likewise partially reversed by anti‐miR‐506‐3p or oe‐ATP7A (Figure 4E,F). Furthermore, Western blot analysis of key cuproptosis‐related proteins showed that LINC02038 knockdown reduced DLAT, FDX1, and LIPT1 protein levels, which were partially restored upon co‐transfection with anti‐miR‐506‐3p or oe‐ATP7A (Figure 4G). These results demonstrate that the LINC02038/miR‐506‐3p/ATP7A axis plays a crucial role in regulating HCC malignant progression and cuproptosis.

Figure 4.

The effect of ATP7A‐associated ceRNA regulatory network in HCC cell malignant progression and cuproptosis. The cells were divided into 6 groups: si‐NC, si‐ LINC02038, si‐ LINC02038+ anti‐miR‐NC, si‐LINC02038+ anti‐miR‐506‐3p, si‐ LINC02038+oe‐NC, si‐ LINC02038+oe‐ATP7A. A. CCK‐8 was employed to detect HCC cell viability. B. Colony formation was employed to detect HCC cell growth. C, D. Transwell was employed to detect HCC cell migration and invasion. E. The intracellular copper levels in transfected HCC cells treated with elesclomol‐Cu (1:1 ratio) for 24 h. F. ROS levels in transfected HCC cells treated with elesclomol‐Cu (1:1 ratio) for 24 h.G. The protein levels of DLAT, FDX1, and LIPT1 in transfected HCC were detected by western blot. ***p < 0.001.

3.5. LINC02038 Promoted HCC Tumorigenesis and Modulated Cuproptosis In Vivo

To further evaluate the oncogenic function of the LINC02038/miR‐506‐3p/ATP7A axis in vivo, we established a stable LINC02038‐knockdown HCC cell line and performed a xenograft tumor assay by subcutaneously injecting these cells into nude mice (Figure 5A). Compared with the control group, tumors in the sh‐LINC02038 group exhibited significantly inhibited growth, as reflected by reduced tumor volume and weight (Figure 5B,C). Subsequent molecular analysis of the harvested tumor tissues revealed that mRNA levels of both LINC02038 and ATP7A were significantly downregulated in the sh‐LINC02038 group, whereas miR‐506‐3p expression increased—consistent with our cellular findings (Figure 5D). Further examination of key cuproptosis regulators showed that protein levels of DLAT, FDX1, and LIPT1 were markedly decreased in tumors with LINC02038 knockdown (Figure 5E). IHC analysis further confirmed reduced ATP7A expression and a lower Ki67 index in the sh‐LINC02038 group compared to the sh‐NC group, aligning with the in vitro mechanistic findings (Figure 5F,G). Taken together, these in vivo findings demonstrated that LINC02038 drove HCC tumor growth, modulated the expression of miR‐506‐3p and ATP7A, and also altered the levels of key regulators of cuproptosis.

Figure 5.

Silencing of LINC02038 inhibits the progression of HCC in vivo. A. Representative images of tumors in nude mice following subcutaneous injection of HCC cells with LINC02038 silencing (sh‐LINC02038) or sh‐NC. B. The tumor growth curve was constructed using the volume data from the xenograft tumors. C. The weight of the tumors was determined. D. The relative expression levels of LINC02038, miR‐506‐3p and ATP7A in xenograft tumors were quantified by qRT‐PCR. E. Protein levels of DLAT, FDX1, and LIPT1 were detected by western blot. F–G. Representative IHC images of ATP7A (F) and Ki67 (G) expression. Original magnification: ×100 and ×200. ***p < 0.001.

4. Discussion

HCC is a malignant tumor characterized by high incidence and mortality [17]. Due to its complex pathogenesis, difficulties in early diagnosis, and limited treatment options, HCC prognosis and clinical management remain serious challenges [18]. Therefore, exploring innovative treatment strategies and therapeutic targets holds significant importance. Database analysis in this study reveals aberrant ATP7A expression in multiple cancers, consistent with previous findings. For example, Shi et al. demonstrated that ATP7A exerts oncogenic functions in gastric cancer, where its high expression indicates poor prognosis and promotes tumor cell proliferation and invasion by modulating the tumor immune microenvironment and related signaling pathways [19]. In contrast, Guan et al. constructed a multifunctional nanoplatform confirming that ATP7A disrupts intracellular copper ion homeostasis, thereby enhancing cuproptosis and ultimately suppressing tumor progression [20].

In this study, we confirmed that ATP7A was significantly upregulated in both HCC tissues and cell lines. Its high expression closely correlated with advanced tumor stage and poor patient prognosis. This expression pattern aligns with previous reports in ovarian cancer, breast cancer, and other malignancies [21, 22], further supporting ATP7A's oncogenic role in HCC. Functionally, ATP7A knockdown effectively inhibited HCC cell proliferation, migration, and invasion, indicating its crucial role in maintaining HCC's malignant phenotype. Furthermore, as a core protein maintaining intracellular copper homeostasis, ATP7A expression directly influenced cellular response to copper metabolism‐related stress. Our research found that ATP7A depletion significantly enhanced HCC cell sensitivity to copper ionophore (elesclomol‐Cu)‐induced cuproptosis. This was evidenced by increased intracellular copper accumulation, elevated ROS levels, and markedly decreased drug IC50 values. These results reveal that ATP7A participated in HCC progression and metastasis by regulating cuproptosis.

To clarify upstream regulatory mechanisms, we identified microRNA‐506‐3p as a direct targeting inhibitor of ATP7A. This miRNA showed low expression in HCC cells, potentially related to its negative regulation of ATP7A. Existing literature reports miR‐506‐3p's tumor‐suppressive function in liver cancer; for example, its overexpression can reverse hepatic stellate cell‐conditioned medium (HSC‐CM)‐induced enhancement of HCC cell invasion, downregulate vimentin expression, and suppress TGF‐β/Smad signaling pathway activity, confirming its tumor‐suppressive role in HCC [23]. Further, we identified long non‐coding RNA LINC02038 as an upstream molecule that competitively binds miR‐506‐3p to regulate its activity. Current research on LINC02038's role in tumors is limited, with only one report indicating it can bind miR‐552‐5p in an m6A modification‐dependent manner, regulate FAM172A expression, and inhibit colorectal cancer progression [24]. This study was the first to construct a complete LINC02038/miR‐506‐3p/ATP7A ceRNA regulatory axis, elucidating its molecular mechanism in influencing cuproptosis and HCC malignant progression by regulating key copper metabolism protein ATP7A. In vivo experiments demonstrated that stable LINC02038 knockdown significantly inhibited xenograft tumor growth. This was accompanied by corresponding expression changes in axis components (downregulated ATP7A, upregulated miR‐506‐3p) and altered levels of downstream cuproptosis‐related proteins, confirming this axis's important tumor‐promoting role in vivo. LINC02038 depletion likely inhibits tumor growth by enhancing tumor cell sensitivity to cuproptosis, providing a novel potential therapeutic target and strategy for HCC treatment.

Certainly, this study has several limitations. First, clinical sample size used remains relatively limited. While initially confirming correlation between ATP7A expression and poor HCC prognosis, its robustness and generalizability as an independent prognostic biomarker require further validation through larger, multi‐center prospective cohort studies. Second, cuproptosis represents a complex biological process involving multi‐level regulation. Although ATP7A knockdown was observed to enhance cellular sensitivity to cuproptosis, precise molecular mechanisms whereby ATP7A regulates expression and function of downstream key proteins (e.g., FDX1, DLAT) and related signaling networks await systematic elucidation. Third, in vivo evidence for the LINC02038/miR‐506‐3p/ATP7A‑cuproptosis axis was incomplete, and rescue experiments and direct cuproptosis readouts were lacking. Therefore, the conclusion that this axis regulates HCC through cuproptosis in vivo requires further experimental support. Finally, research primarily focused on LINC02038/miR‐506‐3p/ATP7A axis function in driving HCC malignant progression. Whether this axis also participates in regulating critical HCC characteristics—such as chemotherapy resistance, targeted therapy sensitivity, and tumor immune microenvironment remodeling—constitutes an important direction for future investigation.

In conclusion, this study delineates the molecular mechanisms by which the LINC02038/miR‐506‐3p/ATP7A axis influenced cuproptosis, thereby driving HCC malignancy and metastasis.

Author Contributions

Zhe Liu: conceptualization, writing – original draft. Weixi Shan: conceptualization, writing – original draft. Wenyu Zhou: data curation, formal analysis. Meiying Long: visualization, validation. Long Yang: conceptualization, writing – review and editing. All authors reviewed the results and approved the final version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: ATP7A overexpression promoted malignant phenotypes of HCC and reduced cellular sensitivity to cuproptosis.

Table S1: The primers used for qRT‐PCR and cell transfection.

Liu Z., Shan W., Zhou W., Long M., and Yang L., “ATP7A Orchestrates Tumor Progression and Cuproptosis in Hepatocellular Carcinoma Via the LINC02038‐miR‐506‐3p Regulatory Circuit,” Journal of Biochemical and Molecular Toxicology 40 (2026): e71027, 10.1002/jbt.71027.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.