ESCC lines KYSE-150 and TE-1 were obtained from the Cell Bank of the Chinese Academy of Sciences. KYSE-150 is one of the most classic and extensively used cell lines in ESCC research, with a well-defined ESCC origin and characteristics. TE-1 is another well-characterized and commonly utilized ESCC cell line. KYSE-150 cultures were maintained in a 1:1 mixture of RPMI-1640 and Ham's F-12 basal media supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. TE-1 cultures were grown in RPMI-1640 containing identical supplements. Cells were incubated at 37°C under a humidified atmosphere containing 5% CO₂ and routinely tested for mycoplasma contamination (negative).

For ectopic expression, the full-length LINC00184 sequence (gene ID, 100302691; National Center for Biotechnology Information) was gene-synthesized and inserted into the pLVX-mCMV-ZsGreen-IRES-Puro lentiviral backbone (Wuhan Viraltherapy Technologies, Co., Ltd.) using conventional restriction-enzyme-based cloning. Nucleic acid constructs were used at a standardized concentration of 2 µg per well for 6-well plate transfection. For transient transfection, logarithmically growing cells were plated at 4×10⁵ cells per well in 6-well plates and transfected with either OE-LINC00184 or empty vector controls (OE-NC) using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Transfection was performed at 37°C for a continuous incubation duration of 48 h under routine cell culture conditions. Total RNA was isolated 48 h post-transfection and LINC00184 overexpression was verified by reverse transcription-quantitative PCR (RT-qPCR) using the primers detailed in Table I. Specifically, total RNA was extracted from transfected cell samples using RNAiso Plus (Takara Bio) as the RNA extraction reagent. Reverse transcription was performed with the PrimeScript^™ RT Reagent Kit (Takara Bio), following the manufacturer's recommended temperature protocols. The cDNA acquired from reverse-transcribed total RNA served as the DNA template for subsequent qPCR analysis. qPCR was conducted using BeyoFast^™ SYBR Green qPCR Mix (Beyotime Biotechnology). GAPDH was applied as the internal reference gene. Standard thermocycling conditions were set as initial denaturation at 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Relative gene expression levels were calculated using the 2^−ΔΔCq method (24). For ectopic expression of NDRG2, the full-length human NDRG2 coding sequence (gene ID, 57447) was synthesized and cloned into the identical lentiviral backbone, generating the overexpression plasmid designated OE-NDRG2. For transient rescue experiments, cells were co-transfected with the specified combinations of OE-LINC00184, OE-NDRG2 or their respective OE-NC using Lipofectamine^® 3000. Successful ectopic expression of NDRG2 was validated at translational levels by western blotting analysis.

In total, three siRNA duplexes targeting distinct regions of LINC00184 (sequences listed in Table II) were chemically synthesized by Sangon Biotech Co., Ltd. KYSE-150 and TE-1 cells were transfected in 6-well plates using Lipofectamine^® RNAiMAX (Thermo Fisher Scientific, Inc.). All siRNA transfections were performed at 37°C following the manufacturer's standard protocol, with a final siRNA concentration of 50 nM. The random non-coding siRNA sequence described below served as the negative control for mouse DNMT1 siRNA interference. Gene silencing efficiency was detected 48 h after transfection, and all subsequent functional experiments were performed at the same 48 h post-transfection time point. The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.). The results of the knock-down efficiency are shown in Fig. S1.

For viability assessment, KYSE-150 and TE-1 cells were seeded at 5×10³ cells per well in 96-well plates, allowed to attach overnight (37°C and 5% CO₂) and then subjected to plasmid transfection or 5-AZA treatment for 48 h. Subsequently, 10 µl of CCK-8 reagent (Dojindo Laboratories, Inc.) were added per well, incubated for 2 h at 37°C, and absorbance at 450 nm was recorded on a SpectraMax^® i3× microplate reader (Molecular Devices, LLC). To determine a functional and non-cytotoxic concentration, the present study performed a dose-optimization experiment using CCK-8 assays across a range of concentrations (0, 0.2, 2, 5, 10 and 20 µM). Based on these results (Fig. S2), the present study selected 2 µM for subsequent functional rescue experiments, as it effectively modulated the phenotype while keeping non-specific cytotoxicity low (cell viability reduction <15%).

KYSE-150 and TE-1 cells were seeded at 5×10⁵ cells per well in 6-well plates and allowed to attach overnight (37°C and 5% CO₂). Following plasmid transfection or 5-AZA exposure (48 h), cells were trypsinized, washed twice with PBS and resuspended in serum-free RPMI-1640 to a density of 3×10⁵ cells/ml. A 200 µl aliquot was plated into the upper compartment of 8.0 µm pore Transwell^® inserts (Corning, Inc.); the lower compartment contained 600 µl complete medium (10% FBS) serving as chemoattractant. After 24 h incubation (37°C and 5% CO₂), non-invading cells on the upper surface were gently removed with cotton swabs. Migratory cells on the underside were fixed in 4% paraformaldehyde for 15 min, stained with 0.5% (w/v) crystal violet for 20 min at room temperature, washed three times with PBS and captured under a phase-contrast microscope. Five random fields per insert were counted by two blinded investigators.

After 48 h post-transfection or 5-AZA exposure, KYSE-150 and TE-1 cells were collected, washed three times with PBS and centrifuged. Apoptosis was then quantified using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (cat. no. C1062S; Beyotime Biotechnology). Briefly, cells were resuspended in 1X binding buffer and stained with 5 µl of FITC-conjugated Annexin V and 10 µl of PI for 15 min in the dark at room temperature, followed by the addition of 2 ml PBS. Apoptosis was evaluated using flow cytometry with a FACScan instrument (BD Biosciences) and analyzed using CellQuest^™ software (version number 5.2.1; BD Biosciences). Unstained control and single-color compensation controls (FITC single-stained and PI single-stained cells) were applied to define negative thresholds and adjust spectral compensation. Firstly, intact single-cell populations were gated according to forward scatter and side scatter parameters to exclude cellular debris and aggregates. Subsequently, quadrant gating was performed on the Annexin V-FITC vs. PI dot plot to distinguish viable cells, early apoptotic cells, late apoptotic cells and necrotic cells for statistical analysis.

To evaluate the methylation status of the NDRG2 promoter, the EpiTect Plus DNA Bisulfite Kit (cat. no. 59124; Qiagen GmbH) was employed. Genomic DNA was extracted from KYSE-150 and TE-1 cells using a conventional DNA isolation method (DP-34; Tiangen Biotech Co., Ltd.). The concentration and purity of extracted genomic DNA were determined via a Nanodrop spectrophotometer. After quantifying the DNA concentration, 1 µg of genomic DNA was subjected to bisulfite treatment following the manufacturer's protocol. Subsequently, the bisulfite-treated DNA was used as a template for PCR amplification, utilizing the primers provided in Table III. The PCR was performed under the following conditions: Initial denaturation at 95°C for 10 min, followed by 35 cycles consisting of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 10 sec, with a final extension step at 72°C for 10 min. The PCR products were analyzed via agarose gel electrophoresis and visualized using an image analysis system. The PCR amplicons were then analyzed by 1% agarose gel electrophoresis and visualized using a Bio-Rad ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.) to assess the methylation status.

When the KYSE-150 cells reached 70–80% confluence, the ChIP assay was performed using a commercial ChIP Kit (cat. no. P2078; Beyotime Biotechnology). The cells were fixed with 1% formaldehyde for 10 min at ambient temperature to cross-link the DNA-protein complexes. Subsequently, the chromatin was fragmented by sonication to generate DNA fragments of 200–1,000 base pairs. The sheared chromatin was then subjected to immunoprecipitation overnight at 4°C using specific antibodies: Mouse anti-DNMT1 (cat. no. ab13537; 1:100; Abcam) and a negative control mouse anti-IgG (cat. no. ab109489; 1:100; Abcam). Protein A/G agarose beads were employed to capture the antibody-bound chromatin complexes. After extensive washing to remove non-specific binding, the cross-links were reversed by incubating the samples at 65°C overnight. The DNA was subsequently purified using phenol/chloroform extraction and ethanol precipitation. To verify the binding of DNMT1 to the NDRG2 promoter, PCR amplification was performed using primers specific to the NDRG2 promoter region. The sequences of these primers are detailed in Table IV. Fluorescent quantitative PCR was performed with the 2^−ΔΔCq calculation. The presence of PCR products indicated the successful enrichment of the NDRG2 promoter region in the immunoprecipitated samples, thereby confirming the interaction between DNMT1 and the NDRG2 promoter.

To elucidate the interaction between LINC00184 and DNMT1, RIP assay was performed in the present study using the RIP kit from EMD Millipore, following the manufacturer's instructions. KYSE-150 cells were disrupted with RIPA lysis buffer (cat. no. P0013B; Beyotime Biotechnology) for 5 min at 4°C. After lysis, the cell lysate was centrifuged at 12,000 × g for 10 min at 4°C to remove cell debris and insoluble impurities. A volume of 100 µl of the lysate was incubated with 50 µl protein A/G magnetic beads (MedChemExpress) conjugated with anti-DNMT1 antibody (cat. no. ab13537; 1:100; Abcam) or control IgG (cat. no. ab109489; 1:100; Abcam) for an extended period at 4°C. The magnetic bead-protein complexes were subsequently isolated using a magnetic separator. The complexes were then treated with proteinase K to release the associated RNA. The purified RNA was analyzed via RT-qPCR. Total RNA extracted from the RIP samples was reverse-transcribed into cDNA using a reverse transcription kit, which was then used as the template for qPCR amplification. The qPCR system included BeyoFast™ SYBR Green qPCR Mix, specific primers targeting the target genes and cDNA template. The thermal cycling conditions were as follows: Pre-denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 30 sec. Relative gene expression levels were calculated using the 2^−ΔΔCq method. The specific primers utilized in this experiment are detailed in Table I.

Protein lysates were obtained from KYSE-150 and TE-1 cells using a cell lysis buffer (cat. no. P0013; Beyotime Biotechnology). Protein concentrations were determined using the Bradford method and subsequently normalized. Equivalent quantities ranging from 20–40 µg of protein were resolved on 12–15% gels using SDS-PAGE and electrotransferred to PVDF membranes (MilliporeSigma). After blocking with 5% BSA for 1 h at ambient temperature, the membranes were probed with primary antibodies overnight at 4°C. The primary antibodies utilized were: Mouse anti-GAPDH (cat. no. 60004-1-Ig; 1:1,000; Proteintech Group, Inc.), rabbit anti-NDRG2 (cat. no. Ab174850; 1:1,000; Abcam), mouse anti-AKT (cat. no. 60203-2-Ig; 1:1,000; Proteintech Group, Inc.), rabbit anti-phosphorylated (p)-AKT (cat. no. 80455-1-RR; 1:1,000; Proteintech Group, Inc.), rabbit anti-PI3K (cat. no. 4292; 1:1,000; CST), and rabbit anti-p-PI3K (cat. no. 17366; 1:1,000; CST), anti-DNMT1 (cat. no. 5032; 1:1,000; CST). The blots were washed with TBST containing 0.1% Tween-20 at room temperature three times for 15 min per wash. The membranes were then incubated with HRP-conjugated secondary antibodies, goat anti-rabbit IgG (cat. no. A0208; 1:5,000; Proteintech Group, Inc.) and goat anti-mouse IgG (cat. no. SA00001-1; 1:5,000; Proteintech Group, Inc.) for 1 h at room temperature. Protein bands were detected using a Clarity^™ Western ECL Substrate (Bio-Rad Laboratories, Inc.) and their relative intensities were assessed by Quantity One analysis tool (Bio-Rad Laboratories, Inc.).

Statistical analyses were conducted using GraphPad Prism software (version 5; Dotmatics). For pairwise comparisons, an unpaired Student's t-test was employed. In cases involving multiple treatment groups, one-way ANOVA was performed, followed by Tukey's post hoc test for multiple comparisons. All experiments in this study were performed with no fewer than three independent biological replicates and the number of experimental repeats varied appropriately according to different experimental designs. Data are presented as mean ± SEM. P<0.05 was considered to indicate a statistically significant difference.

In order to explore the role of LINC00184 in ESCC cell proliferation, migration and apoptosis, KYSE-150 and TE-1 cells were transfected with the OE-LINC00184 plasmid to overexpress LINC00184. The expression level of LINC00184 in KYSE-150 and TE-1 cells following transfection was verified using RT-qPCR. The results demonstrated that the expression level of LINC00184 was significantly increased after transfection with the OE-LINC00184 plasmid in KYSE-150 (Fig. 1A) and TE-1 cells (Fig. 1B). In addition, the biological behaviors of cells were assessed using CCK-8 staining, Transwell and apoptosis assays. As presented in Fig. 1C and D, overexpression of LINC00184 markedly enhanced the cell viability of these two cells. The Transwell assay results revealed the increased number of migrated cells in the OE-LINC00184 groups compared with the control groups which means that overexpression of LINC00184 significantly promoted the migration of KYSE-150 cells (Fig. 1E and F) and TE-1 cells (Fig. 1G and H). Additionally, flow cytometry analysis of apoptosis indicated that the apoptotic rates in the OE-LINC00184 groups were significantly lower compared with those in the control groups which means that overexpression of LINC00184 reduced the apoptosis rate in both KYSE-150 cells (Fig. 1I and J) and TE-1 cells (Fig. 1K and L). In summary, these findings suggested that overexpression of LINC00184 serves a key role in promoting the proliferation, migration, and inhibiting apoptosis of ESCC cells.

To elucidate the molecular mechanisms underlying the effects of LINC00184 in ESCC cells, the present study focused on its impact on NDRG2 expression and the PI3K/AKT signaling pathway. NDRG2 is a tumor suppressor protein that inhibits cell proliferation and promotes apoptosis in various cancer types, including lung, liver and pancreatic cancer (16–18). NDRG2 can inhibit the PI3K/AKT pathway by acting as a bridge between PP2A and PTEN, leading to the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate and subsequent inhibition of AKT activity (21). Due to this background, the present study hypothesized that LINC00184 might influence ESCC cell behavior by regulating NDRG2, thereby affecting the PI3K/AKT pathway. Western blotting analysis in KYSE-150 and TE-1 cells revealed that overexpression of LINC00184 significantly decreased NDRG2 protein levels (Fig. 2A, B, F and G). Consistent with this, RT-qPCR analysis confirmed that LINC00184 overexpression also led to a significant reduction in NDRG2 mRNA levels in both cell lines (Fig. 2E and J), demonstrating transcriptional downregulation. Concurrently, the overexpression increased the phosphorylation of AKT (Fig. 2A, C, F and H) and PI3K (Fig. 2A, D, F and I), indicative of PI3K/AKT pathway activation. These results suggested that LINC00184 may promote ESCC cell proliferation and survival by transcriptionally repressing NDRG2 and leading to reduced NDRG2 protein expression, thereby influencing the PI3K/AKT pathway.

To directly establish the causal role of NDRG2 downregulation in mediating the effects of LINC00184, the present study performed a rescue experiment by re-expressing NDRG2 in TE-1 cells stably overexpressing LINC00184. Western blotting analysis confirmed the successful restoration of NDRG2 protein (Figs. 3A and S3A). Notably, this restoration significantly reversed the LINC00184-induced hyperphosphorylation of AKT (Figs. 3A and S3B), indicating that NDRG2 reconstitution effectively suppressed the activation of the PI3K/AKT pathway. Correspondingly, functional assays demonstrated that re-expression of NDRG2 significantly attenuated the enhanced cell viability promoted by LINC00184 overexpression (Fig. 3B). Collectively, these data provided direct genetic evidence that the downregulation of NDRG2 is a pivotal and causative event through which LINC00184 activates the PI3K/AKT pathway and drives proliferative advantage in ESCC cells.

To elucidate the regulatory mechanisms of LINC00184 on NDRG2 expression in ESCC cells, the present study first validated the effects of both LINC00184 overexpression and knockdown using siRNA (Fig. 4A and B). The present study identified that overexpression of LINC00184 markedly increased the methylation level of the NDRG2 promoter, as confirmed by MSP assay (Fig. 4C and D). By contrast, silencing LINC00184 using siRNA markedly reduced the methylation level of the NDRG2 promoter (Fig. 4C and D). These initial findings prompted further investigation into the role of DNA methylation in LINC00184-mediated NDRG2 regulation. DNMT1 is a key enzyme responsible for maintaining DNA methylation patterns, particularly during DNA replication (13). Due to its key function in epigenetic regulation, the present study hypothesized that LINC00184 might influence NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. To investigate this hypothesis, the present study conducted ChIP assays to examine the enrichment of DNMT1 at the NDRG2 promoter region. The results indicated that overexpression of LINC00184 led to significantly increased enrichment of DNMT1 at the NDRG2 promoter (Fig. 4E), suggesting that LINC00184 recruits DNMT1 to the NDRG2 promoter. By contrast, silencing LINC00184 using siRNA significantly decreased the enrichment of DNMT1 at the NDRG2 promoter (Fig. 4E), further supporting the role of LINC00184 in DNMT1 recruitment. To confirm the direct interaction between LINC00184 and DNMT1, the present study performed RIP assays. The results revealed that overexpression of LINC00184 significantly increased the enrichment of LINC00184 by DNMT1 (Fig. 4F and G), indicating a direct interaction between LINC00184 and DNMT1. By contrast, silencing LINC00184 significantly decreased this enrichment (Fig. 4F and G), reinforcing the notion that LINC00184 directly interacts with DNMT1. To further confirm the role of DNMT1 in LINC00184-mediated NDRG2 suppression, ESCC cells overexpressing LINC00184 were treated with the DNMT1 inhibitor 5-AZA. The results demonstrated that 5-AZA treatment effectively reversed the LINC00184-induced methylation of the NDRG2 promoter (Fig. 4H and I) and restored NDRG2 expression (Fig. 4J-M). This indicated that the methylation changes induced by LINC00184 are reversible and that DNMT1 activity is a key factor in this process. To establish a more specific causal association between DNMT1 activity and this regulatory axis, the present study employed siRNA to directly knock down DNMT1 in LINC00184-overexpressing cells. This genetic intervention successfully reversed the LINC00184-induced downregulation of NDRG2 protein (Figs. 4N and S4A), mirroring the effect observed with the pharmacological inhibitor 5-AZA. Notably, to definitively rule out the alternative possibility that LINC00184 suppresses NDRG2 by first upregulating DNMT1 expression, the present study assessed DNMT1 levels following LINC00184 manipulation. Neither DNMT1 mRNA (Fig. 4O) nor total protein levels (Figs. 4P and S4B) were significantly altered by LINC00184 overexpression. This key control experiment confirmed that LINC00184 does not affect DNMT1 abundance but instead acts by modulating the functional recruitment and activity of pre-existing DNMT1 to epigenetically silence the NDRG2 promoter. Therefore, the convergent evidence from pharmacological (5-AZA) and genetic (siDNMT1) inhibition, coupled with the unchanged DNMT1 expression profile, solidifies DNMT1 as the key epigenetic effector downstream of LINC00184.

Having established that LINC00184 recruits DNMT1 to silence NDRG2, the present study then investigated whether this epigenetic mechanism is responsible for the downstream activation of the oncogenic PI3K/AKT pathway. The present study first confirmed that pharmacological inhibition of DNMT1 with 5-AZA in LINC00184-overexpressing cells effectively reversed the hyperphosphorylation of both PI3K and AKT (Fig. 5A-F). To establish a specific genetic association, siRNA-mediated knockdown of DNMT1 was performed. Consistent with the pharmacological data, siDNMT1 significantly attenuated the LINC00184-induced increase in AKT phosphorylation (Fig. 5G and H). This convergence of evidence from independent inhibitory strategies definitively confirmed that the DNMT1-mediated epigenetic silencing of NDRG2 is the key mechanistic event through which LINC00184 activates the PI3K/AKT pathway in ESCC cells.

To determine whether the DNMT1-mediated epigenetic axis is functionally required for the oncogenic phenotypes driven by LINC00184, the present study inhibited DNMT1 activity using both pharmacological and genetic approaches. Treatment of LINC00184-overexpressing cells with the DNMT1 inhibitor 5-AZA significantly reversed the pro-tumorigenic phenotypes: It significantly reduced cell viability (Fig. 6A and F), significantly attenuated enhanced cell migration (Fig. 6B, D, G and I) and significantly increased the apoptotic rate (Fig. 6C, E, H and J) back towards control levels. To provide specific genetic confirmation, siRNA-mediated knockdown of DNMT1 (siDNMT1) was performed in the same context. Consistent with the pharmacological inhibition, genetic depletion of DNMT1 also significantly reduced the enhanced cell viability induced by LINC00184 overexpression (Fig. 6K).

Collectively, these results demonstrated that DNMT1 activity is key to executing the full spectrum of the oncogenic functions of LINC00184, including promoting proliferation, migration and suppressing apoptosis. The concordance between chemical and genetic inhibition solidifies DNMT1 as a key downstream effector and a potential therapeutic target within this pathway.